Optical analysis device, optical analysis method and computer program for optical analysis using single particle detection

Abstract

In the optical analysis technique of detecting an existence of a single particle in a sample solution with a confocal microscope or a multiphoton microscope according to the scanning molecule counting method of the present invention, the position of a light detection region is moved in the sample solution; the light intensity from the light detection region is measured so that light intensity data will be generated; a first occurrence probability in assuming a first condition that no single particles exist in the light detection region and a second occurrence probability in assuming a second condition that a single particle exists in the light detection region for a time variation of light intensity value on the light intensity data are computed; and a signal indicating each single particle is detected based on those occurrence probabilities, and thereby enabling improvements in the sensitivity and/or S/N ratio.

The invention claimed is: 
     
       1. An optical analysis device which detects a single particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, comprising:
 a light detection region mover which moves a position of a light detection region of the optical system in the sample solution; 
 a light detector which detects light from the light detection region; and 
 a signal processor which generates time series light intensity data of the light from the light detection region detected with the light detector during the moving of the position of the light detection region in the sample solution and detects a signal indicating an existence of each single particle individually in the time series light intensity data; 
 wherein the signal processor computes a first occurrence probability in assuming a first condition that no single particles exist in the light detection region and a second occurrence probability in assuming a second condition that a single particle exists in the light detection region for a time variation of light intensity value in each analysis window set out in time series on the time series light intensity data; and detects a signal indicating an existence of each single particle on the time series light intensity data based on the first and second occurrence probabilities. 
 
     
     
       2. The device of  claim 1 , wherein the single particle has a predetermined characteristic value; the light detector detects separately at least two mutually different components of the light from the light detection region; the signal processor generates time series light intensity data of each of the components; the signal processor further computes the first and second occurrence probabilities of each of the components; the second occurrence probability of each of the components is a function of the predetermined characteristic value; and a signal indicating an existence of the single particle having the predetermined characteristic value on the time series light intensity data is detected based on the first and second occurrence probabilities of each of the components. 
     
     
       3. The device of  claim 2 , wherein the single particles include single particles of two or more kinds which have mutually different predetermined characteristic values; the second occurrence probability of each of the components which is a function of the mutually different predetermined characteristic value is computed for each of the kinds of the single particles; a signal indicating an existence of the single particle on the time series light intensity data is detected for each of the kinds of the single particles based on the first occurrence probability of each of the components and the second occurrence probability of each of the components for each of the two or more kinds of the single particles. 
     
     
       4. The device of  claim 2 , wherein the single particle is a light-emitting particle; the signal indicating an existence of each single particle is a temporary increase of the light intensity; and the predetermined characteristic value is a polarization anisotropy of the single particle. 
     
     
       5. The device of  claim 2 , wherein the single particle is a light-emitting particle; the signal indicating an existence of each single particle is a temporary increase of the light intensity; and the predetermined characteristic value is a ratio of emitted light intensities in mutually different emission wavelength bands of the single particle. 
     
     
       6. The device of  claim 1 , wherein the single particle is a non light-emitting particle; the light from the light detection region includes background light; and the signal indicating an existence of each single particle is a temporary reduction of the light intensity from the background light. 
     
     
       7. An optical analysis method of detecting a single particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, comprising steps of:
 moving a position of a light detection region of the optical system in the sample solution; 
 detecting light from the light detection region during the moving of the light detection region in the sample solution to generate time series light intensity data; 
 computing a first occurrence probability in assuming a first condition that no single particles exist in the light detection region and a second occurrence probability in assuming a second condition that a single particle exists in the light detection region for a time variation of light intensity value in each analysis window set out in time series on the time series light intensity data; and 
 detecting a signal indicating an existence of each single particle on the time series light intensity data based on the first and second occurrence probabilities. 
 
     
     
       8. The method of  claim 7 , wherein the first and second occurrence probabilities are computed based upon a light intensity value in each unit time in the analysis window and an expected value in each unit time in assuming the first and second conditions, respectively. 
     
     
       9. The method of  claim 8 , wherein an unit time occurrence probability of the light intensity value in each unit time is computed under an assumption that the light intensity value in each unit time follows Poisson distribution having the expected value in the each unit time, and the first and second occurrence probabilities are computed using the corresponding unit time occurrence probabilities, respectively. 
     
     
       10. The method of  claim 7 , wherein the single particle has a predetermined characteristic value; at least two mutually different components of the light from the light detection region are detected separately; time series light intensity data of each of the components are generated; the first and second occurrence probabilities of each of the components are further computed; the second occurrence probability of each of the components is a function of the predetermined characteristic value; and a signal indicating an existence of the single particle having the predetermined characteristic value on the time series light intensity data is detected based on the first and second occurrence probabilities of each of the components. 
     
     
       11. The method of  claim 10 , wherein the single particles include single particles of two or more kinds which have mutually different predetermined characteristic values; the second occurrence probability of each of the components which is a function of the mutually different predetermined characteristic value is computed for each of the kinds of the single particles; a signal indicating an existence of the single particle on the time series light intensity data is detected for each of the kinds of the single particles based on the first occurrence probability of each of the components and the second occurrence probability of each of the components for each of the two or more kinds of the single particles. 
     
     
       12. The method of  claim 10 , wherein the single particle is a light-emitting particle; the signal indicating an existence of each single particle is a temporary increase of the light intensity; and the predetermined characteristic value is a polarization anisotropy of the single particle. 
     
     
       13. The method of  claim 10 , wherein the single particle is a light-emitting particle; and the signal indicating an existence of each single particle is a temporary increase of the light intensity; the predetermined characteristic value is a ratio of emitted light intensities in mutually different emission wavelength bands of the single particle. 
     
     
       14. The method of  claim 7 , wherein the single particle is a non light-emitting particle; the light from the light detection region includes background light; and the signal indicating an existence of each single particle is a temporary reduction of the light intensity from the background light. 
     
     
       15. A non-transitory computer readable storage device having a computer program product including programmed instructions for optical analysis of detecting light from a light-emitting particle dispersed and moving at random in a sample solution using an optical system of a confocal microscope or a multiphoton microscope, said programmed instructions causing a computer to perform steps comprising:
 moving a position of a light detection region of the optical system in the sample solution; 
 detecting light from the light detection region during the moving of the light detection region in the sample solution to generate time series light intensity data; 
 computing a first occurrence probability in assuming a first condition that no single particles exist in the light detection region and a second occurrence probability in assuming a second condition that a single particle exists in the light detection region for a time variation of light intensity value in each analysis window set out in time series on the time series light intensity data; and 
 detecting a signal indicating an existence of each single particle on the time series light intensity data based on the first and second occurrence probabilities. 
 
     
     
       16. The non-transitory computer readable storage device of  claim 15 , wherein the single particle has a predetermined characteristic value; at least two mutually different components of the light from the light detection region are detected separately; time series light intensity data of each of the components are generated; the first and second occurrence probabilities of each of the components are further computed; the second occurrence probability of each of the components is a function of the predetermined characteristic value; and a signal indicating an existence of the single particle having the predetermined characteristic value on the time series light intensity data is detected based on the first and second occurrence probabilities of each of the components. 
     
     
       17. The non-transitory computer readable storage device of  claim 16 , wherein the single particles include single particles of two or more kinds which have mutually different predetermined characteristic values; the second occurrence probability of each of the components which is a function of the mutually different predetermined characteristic value is computed for each of the kinds of the single particles; a signal indicating an existence of the single particle on the time series light intensity data is detected for each of the kinds of the single particles based on the first occurrence probability of each of the components and the second occurrence probability of each of the components for each of the two or more kinds of the single particles. 
     
     
       18. The non-transitory computer readable storage device of  claim 16 , wherein the single particle is a light-emitting particle; the signal indicating an existence of each single particle is a temporary increase of the light intensity; and the predetermined characteristic value is a polarization anisotropy of the single particle. 
     
     
       19. The non-transitory computer readable storage device of  claim 16 , wherein the single particle is a light-emitting particle; the signal indicating an existence of each single particle is a temporary increase of the light intensity; and the predetermined characteristic value is a ratio of emitted light intensities in mutually different emission wavelength bands of the single particle. 
     
     
       20. The non-transitory computer readable storage device of  claim 15 , wherein the single particle is a non light-emitting particle; the light from the light detection region includes background light; and the signal indicating an existence of each single particle is a temporary reduction of the light intensity from the background light.